Outlook

The vast variety of available methodologies and constructs have not been systematically compared for their efficiency. This is mainly

Table 2

Human Single-Pot Phage Display Libraries

Library vector

Library type

Antibody type fdDOG-

21ox,pUC19-21ox fdTet pAALFab pAP-IIIg scFv pCANTAB6

pCESl pCombS

pCombS pDAN5

pDN322

Semisynthetic

Naive scFv

Semisynthetic (anti-hen egg white lysozyme Ab framework) Fab

Naive scFv

Naive scFv

Naive Fab Semisynthetic (antitetanus Ab framework2) Fab Semisynthetic (antitetanus Ab framework2) Fab Naive scFv

Semisynthetic (VH DP47

and VL DPK22 V-genes) scFv

Library cloning Library strategy size Reference

PCR with random CDR3 Primers, Cre-lox Recloning of a naive library1 PCR with random CDR Primers, assembly PCR Assembly PCR Assembly PCR Three-step cloning (Lchain, VH) " PCR with random CDR H3 Primers PCR with random CDR H3 Primers Cre-lox

Random CDR3 Primer, assembly PCR

5 x 10s 46

5 xlO7 103

>108 104

3 x10n 16

3 x 10s 82

pDNEK (ETH2

library) Semisynthetic (germline pEXmide5

pFAB5c-His

(n-CoDeR library) pHENl pHENl pHENl

Co pHENl-VX3 pHENl-VX3

pHENl-Vk3 pHEN2 (Griffin 1 library)

Semisynthetic (VH

DP47, VI DPL16 and Vk

DPK 22 V-genes) scFv

VH-DP47 and VL-DPL3

framework) scFv

Semisynthetic (germline scFv VH-DP47 and VL-DPL3

framework) scFv

Naive scFv

Naive scFv

Naive scFv Semisynthetic (VX3 anti-

BSA Ab light chain) scFv Semisynthetic (VX3 anti-

BSA Ab light chain) scFV

Semisynthetic (VH)/

naive (VL) scFv

Semisynthetic scFv

Random CDR3 Primer, assembly PCR

Assembly PCR,

CDR shuffling Assembly PCR, Assembly PCR

CDR shuffling Assembly PCR Assembly PCR

Assembly PCR PCR with random

CDR H3 Primers PCR with random CDR H3 Primers Three-step cloning, PCR with random CDR H3 Primers Recloning of the lox library in scFv format3

5 x 10s

9x 106

107x 10s 15

107 92

>108 107

www.mrc-1.2 x 109 cpe.cam.ac.uk

(continued)

Table 2 (Continued)

Human Single-Pot Phage Display Libraries

Table 2 (Continued)

Human Single-Pot Phage Display Libraries

Antibody

Library cloning

Library

Library vector

Library type

type

strategy

size

Reference

PCR with random

1.47 x 108

pIT2 (Tom I/ J

Semisynthetic (3x VH

CDR2 and CDR3

/1.37 x 108

97

library)

and 4x Vk V-genes) Semisynthetic (anti-

scFv

Primers PCR with random CDR Primers, 2

pLG18

HER2 Ab framework)

Fab

step cloning Two-step cloning

2-3 x 108

98

pMorph series

+ 2 step CDR3

(HuCAL library)

Synthetic

scFv

replacement

2 x 109

100

pScUAGDcp3

Semisynthetic

scFv connected to

Three-step cloning with random CDR3

Ck

Primers

1.7 x 107

101

scFv (with N-

terminus of

pSEX81

Naive

CH1 and CL) scFv (with N-terminus of

Two-step cloning

4 x 107

109

pSEX81

Naive

CH1 and CL) scFv (with N-terminus of

1.6 x 107/1.8 x

110

pSEX81

Naive

CH1 and CL)

Four-step cloning

107/4 x 107

111

owing to the fact that experimental strategies allowing evaluation of the performance of any given vector other than using it for library construction and panning, is hard to formulate. The proof of the pudding is in the eating so, we have to analyze the resulting libraries produced by the various methods. However, this is not an easy task because different methods are used for each type of panning. When comparing successfully used human "single-pot" libraries (Table 2), it is evident that most of them use scFv fragments, but this is because the construction of scFv phage display vectors is less complicated compared to Fab phage display libraries. More fine tuning is necessary when expressing two chains in the case of Fab fragments, because the ratio of light chain to heavy chain must be harmonized, as in the case of scFv fragments.

Various leader sequences, promoters, and cloning strategies have been successfully used, with none of them providing a perfect construct. In conclusion, there are many different ways to success with many factors that can be altered depending on individual applications. Two factors are probably more important than the others and these are the careful control of phage biology during the panning process and secondly the use of an initial antibody library as large as possible.

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